DE10225593A1 - Increasing the Vitamin E content of plants, useful e.g. in foods and animal feeds, by transgenic expression of tocopherol synthesis enhancing protein-1 - Google Patents

Abstract

Increasing the Vitamin E content of plants, or their tissues, organs, parts, cells or replicative products, comprises transgenic expression of a protein (2) of 151 amino acids (sequence reproduced) or its functional equivalent with at least 60% identity. Independent claims are also included for the following: (1) transgenic expression construct (EC) comprising a sequence (A) that encodes (2), or its functional equivalent, under control of a promoter functional in plants; (2) transgenic expression vector that includes EC; and (3) transgenic plants (or their tissues, organs, parts, cells or replicative products) that contain EC or the vector of item (2).

Description

The invention relates to transgenic expression constructs and Process for increasing the vitamin E content in vegetable Organisms, preferably in algae, by transgenic expression of ORF sll0832 from Synechocystis sp. PCC6803. The invention relates also transgenic expression constructs for the expression of ORF sll0832 in plant organisms, preferably in algae, transgenic plant organisms expressing ORF sll0832, as well as the Use of said transgenic plant organisms for Manufacture of food, animal feed, seeds, pharmaceuticals or fine chemicals, especially for the production of vitamin E.

The eight naturally occurring compounds with vitamin E activity are derivatives of 6-chromanol (Ullmann's Encyclopedia of Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft, Chapter 4., 478-488, vitamin E). The group of tocopherols (1a-d) has a saturated side chain, the group of tocotrienols (2a-d) has an unsaturated side chain:


1a, α-tocopherol: R ¹ = R ² = R ³ = CH ₃
1b, β-tocopherol [148-03-8]: R ¹ = R ³ = CH ₃ , R ² = H
1c, γ-tocopherol [54-28-4]: R ¹ = H, R ² = R ³ = CH ₃
1d, δ-tocopherol [119-13-1]: R ¹ = R ² = H, R ³ = CH ₃

2a, α-tocotrienol [1721-51-3]: R ¹ = R ² = R ³ = CH ₃
2b, β-tocotrienol [490-23-3]: R ¹ = R ³ = CH ₃ , R ² = H
2c, γ-tocotrienol [14101-61-2]: R ¹ = H, R ² = R ³ = CH ₃
2d, δ-tocotrienol [25612-59-3]: R ¹ = R ² = H, R ³ = CH ₃

In the present invention, vitamin E includes all eight tocopherols and tocotrienols with vitamin E Activity understood.

Vitamin E compounds have a high economic value as additives in the food and feed sector, in pharmaceuticals Formulations and in cosmetic applications.

There are attempts to increase the metabolic flow Increase in tocopherol or tocotrienol content in transgenic plants by overexpression of individual Tocopherol biosynthesis genes (WO 97/27285; WO 99/04622; WO 99/23232; WO 00/10380; Shintani and Dellapenna, Science 282 (5396): 2098-2100, 1998; Tsegaye et al. Annual meeting of the American Society of Plant Physiologists (July 24-28, 1999, Baltimore, USA) Abstract No. 413).

Synechocystis sp. PCC 6803 is a unicellular, not nitrogen fixing cyanobacterium that has been genetically well studied (Churin et al. (1995) J Bacteriol 177: 3337-3343), easy can be transformed (Williams (1988) Methods Enzymol 167: 766-778) and a very active homologue Has recombination ability. The PCC 6803 strain was created in 1968 by R. Kunisawa as Aphanocapsa N-1 in California, USA, isolated from fresh water and is today about the "Pasteur Culture Collection of Axenic Cyanobacterial Strains "(PCC), Unité de Physiologie Microbienne, Paris, France. The overall genomic sequence of Synechocystis sp. PCC 6803 was published from 1995 (Kaneko et al. (1995) DNA Research 2: 153-166; Kaneko et al. (1995) DNA Research 2: 191-198; Kaneko et al. (1996) DNA Research 3: 109-136; Kaneko et al. (1996) DNA Research 3: 185-209; Kaneko and Tabata (1997) Plant Cell Physiol 38: 1171-1176; Kotani and Tabata (1998) Annu Rev Plant Physiol 49: 151-171) and is on the Internet (http: / / www.kazusa.or.jp/cyano/cyano.html) under the name "CyanoBase" released. Effective expression systems for Synechocystis 6803 are described in the literature (Mermet- Bouvier et al. (1993) Curr Microbiol 27: 323-327; Mermet-Bouvier and Chauvat (1993) Curr Microbiol 28: 145-148; Murphy and Stevens (1992) Appl Environ Microbiol 58: 1650-1655; Takeshima et al. (1994) Proc Natl Acad Sci USA 91: 9685-9689; Xiaoqiang et al. (1997) Appl Environ Microbiol 63: 4971-4975; Ren et al. (1998) FEMS Microbiol Lett 158: 127-132).

Despite some success, there is still a need for one Optimization of vitamin E biosynthesis. The object of the invention was to achieve this to provide additional procedures that the Increase vitamin E biosynthesis and thus beneficial lead transgenic plant organisms.

This object is achieved by the present invention. in the In the context of the invention, the protein was encoded by ORF sll0832 from Synechocystis sp. PCC 6803 (GenBank Acc.-No .: NP_442444; gi: 16331716; SEQ ID NO: 2) surprisingly as Key factor in vitamin E biosynthesis identified (as a result of Tocen-1 for "Tocopherol synthesis enhancing protein" -1). Tocen-1 is in the GenBank as an unknown protein without any Function annotation classified. The protein has no significant Homologies to other proteins of known function. Surprisingly, it was found that the transgenic expression of this protein, the vitamin E content in plant organisms can increase.

• a) transgene Expression des Tocen-1 Proteins kodiert durch SEQ ID NO: 2 oder eines funktionellen Äquivalentes desselben oder eines funktionell äquivalenten Teils der vorgenannten in dem besagten pflanzlichen Organismus oder einem Gewebe, Organ, Teil oder Zelle des besagten pflanzlichen Organismus, und
• b) Auswahl von pflanzlichen Organismen, bei denen - im Unterschied oder Vergleich zum Ausgangsorganismus - der Vitamin E- Gehalt in dem besagten pflanzlichen Organismus oder einem Gewebe, Organ, Teil oder Zelle des besagten pflanzlichen Organismus erhöht ist.

A first object of the invention comprises methods for increasing the vitamin E content in plant organisms

• a) transgenic expression of the Tocen-1 protein encoded by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the aforesaid in said plant organism or a tissue, organ, part or cell of said plant organism, and
• b) Selection of plant organisms in which - in contrast to or compared to the starting organism - the vitamin E content in said plant organism or in a tissue, organ, part or cell of said plant organism is increased.

In principle, the method according to the invention can be applied to all plant organisms are used, preferably on those produce vitamin E naturally, especially preferred to those for industrial production of natural Vitamin E can be used.

Another object of the invention relates to transgenic Expression constructs for the expression of a nucleic acid sequence encoding the Tocen-1 protein encoded by SEQ ID NO: 2 or a functional equivalent of the same or one functionally equivalent part of the aforementioned. Especially the sequence according to SEQ ID NO: 1 and that based on the degeneration of the genetic code Sequences and functionally equivalent parts of the aforementioned.

"Plant organism or cells derived from it" generally means every cell, tissue, part or propagation material (like seeds or fruits) of an organism used for photosynthesis is capable. All are included within the scope of the invention Genera and species of higher and lower plants of the Plant Kingdom. Annuals, perennials, monocotyledons and dicotyledons Plants are preferred. Included are mature plant, Seeds, shoots and seedlings, as well as parts derived from them, Propagation material (for example tubers, seeds or fruits) and Cultures, for example cell or callus cultures.

"Plant" in the context of the invention means all genera and Species of higher and lower plants in the plant kingdom. Included under the term are the mature plants, Seeds, sprouts and seedlings, as well as parts derived from them, Propagation material, plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures, as well as all others Types of groupings from plant cells to functional or structural units. Mature plants mean plants to everyone any stage of development beyond the seedling. seedling means a young, immature plant in an early Development.

"Plant" includes all annual and perennial, monocot and dicot plants and closes exemplary but not restrictive of those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea and Populus on.

Plants from the following plant families are preferred: Amaranthaceae, Asteraceae, Brassicaceae, Caryphyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.

Preferred monocot plants are selected in particular from the monocotyledonous crops, such as the Gramineae such as rice, corn, wheat or other cereals such as Barley, millet, rye, triticale or oats and sugar cane as well as all types of grasses.

• - Asteraceae wie Sonnenblume, Tagetes oder Calendula und andere mehr,
• - Compositae, besonders die Gattung Lactuca, ganz besonders die Art sativa (Salat) und andere mehr,
• - Cruciferae, besonders die Gattung Brassica, ganz besonders die Arten napus (Raps), campestris (Rübe), oleracea (z. B. Kohl, Blumenkohl oder Broccoli und weitere Kohlarten); und der Gattung Arabidopsis, ganz besonders die Art thaliana sowie Kresse oder Canola und andere mehr,
• - Cucurbitaceae wie Melone, Kürbis oder Zucchini und andere mehr,
• - Leguminosae besonders die Gattung Glycine, ganz besonders die Art max (Sojabohne) Soja sowie Alfalfa, Erbse, Bohnengewächsen oder Erdnuss und andere mehr
• - Rubiaceae, bevorzugt der Unterklasse Lamiidae wie beispielsweise Coffea arabica oder Coffea liberica (Kaffeestrauch) und andere mehr,
• - Solanaceae besonders die Gattung Lycopersicon, ganz besonders die Art esculentum (Tomate) und die Gattung Solanum, ganz besonders die Art tuberosum (Kartoffel) und melongena (Aubergine) und die Gattung Capsicum, ganz besonders die Art annum (Pfeffer), sowie Tabak oder Paprika und andere mehr,
• - Sterculiaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Theobroma cacao (Kakaostrauch) und andere mehr,
• - Theaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Camellia sinensis oder Thea sinensis (Teestrauch) und andere mehr,
• - Umbelliferae, besonders die Gattung Daucus (ganz besonders die Art carota (Karotte)) und Apium (ganz besonders die Art graveolens dulce (Selarie)) und andere mehr;

sowie Lein, Soja, Baumwolle, Hanf, Flachs, Gurke, Spinat, Möhre, Zuckerrübe und den verschiedenen Baum-, Nuss- und Weinarten, insbesondere Banane und Kiwi. The invention is very particularly preferably applied from dicotyledonous plant organisms. Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example

• - Asteraceae such as sunflower, tagetes or calendula and others,
• - Compositae, especially the genus Lactuca, especially the species sativa (lettuce) and others,
• - Cruciferae, especially the genus Brassica, especially the species napus (rape), campestris (turnip), oleracea (e.g. cabbage, cauliflower or broccoli and other types of cabbage); and the genus Arabidopsis, especially the species thaliana as well as cress or canola and others,
• - Cucurbitaceae such as melon, pumpkin or zucchini and others,
• - Leguminosae especially the genus Glycine, especially the type max (soybean) soy, as well as alfalfa, peas, beans or peanuts and others
• Rubiaceae, preferably of the subclass Lamiidae such as Coffea arabica or Coffea liberica (coffee bush) and others,
• - Solanaceae especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant) and the genus Capsicum, especially the species annum (pepper), as well as tobacco or Peppers and others,
• Sterculiaceae, preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) and others,
• Theaceae, preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others,
• - Umbelliferae, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (selaria)) and others;

as well as flax, soybeans, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various types of trees, nuts and wines, especially banana and kiwi.

Also includes ornamental plants, useful or ornamental trees, Flowers, cut flowers, shrubs or lawn. Exemplary, however non-restrictive angiosperms, bryophytes such as Hepaticae (liverwort) and Musci (moss); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms like conifers, cycads, ginkgo and gnetals, the families the rosaceae like rose, ericaceae like rhododendrons and azaleas, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae like cloves, Solanaceae like petunias, Gesneriaceae like that African violets, Balsaminaceae such as balsam, Orchidaceae like orchids, iridaceae like gladiolus, iris, freesia and crocus, Compositae like marigold, Geraniaceae like geranium, Liliaceae like the dragon tree, Moraceae like Ficus, Araceae like Philodendron and others.

Plant organisms in the sense of the invention are still other photosynthetically active competent organisms, such as Example algae, cyanobacteria and mosses. Preferred algae are Green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. In particular Synechocystis is preferred.

Most preferred are plant organisms that contribute to the vitamin E-production are suitable, such as rapeseed, sunflower, Sesame, safflower, olive tree, soybean, corn, wheat or various Types of nuts such as walnut or almond.

"Vitamin E content" means the sum of tocopherols and Tocotrienols in a plant organism or tissue, Organ, part or cell of the same. These include tocopherols and tocotrienols prefer the 8 compounds described above α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrieno, γ-tocotrienol and δ-tocotrienol. Especially the vitamin E content in the seed of a vegetable is preferred Organism increased.

"Increasing" the vitamin E content means increasing the Vitamin E content in a plant organism or a part, tissue or organ thereof, preferably in the Organs of the same. The vitamin E content is compared to a not subject to the inventive method, but otherwise unchanged starting plant under otherwise the same Framework conditions by at least 5%, preferably at least 10%, particularly preferably at least 15%, very particularly preferably at least 20%, most preferably at least 25% increased. Framework conditions mean everyone for germination, cultivation or growth of the plant relevant conditions we soil, Climate or light conditions, fertilization, irrigation, Plant protection measures etc.

Functional equivalents mean in particular natural or artificial mutations of the Tocen-1 protein according to SEQ ID NO: 2 as well as homologous polypeptides from other organisms, preferably from herbal organisms that are the same essential Have properties.

• a) ein Molekulargewicht in einem Bereich von ungefähr 10 bis ungefähr 25 kDa, bevorzugt ungefähr 15 bis ungefähr 20 kDa, besonders bevorzugt ungefähr 17 kDa
• b) einen theoretischen isoelektrischen Punkt in einem Bereich von ungefähr 8 bis ungefähr 9,5, bevorzugt ungefähr 8,4 bis ungefähr 9,1, besonders bevorzugt ungefähr pH 8,7
• c) einen sauren Charakter bevorzugt mit einer Gesamtnettoladung bei neutralem pH in einem Bereich von ungefähr 2 bis ungefähr 3, bevorzugt ungefähr 2,2 bis ungefähr 2,8, besonders bevorzugt ungefähr 2,6,
• d) eine helikale Sekundärstruktur gemäß computergestützter Vorhersage beispielsweise mit dem Programm zur Sekundärstrukturvorhersagen in GenomMax v.3.1 (InforMax, Inc.) basierend auf den Arbeiten von Qian (Qian N and Sejnowski T (1988) J Mol Biol 202: 865-884) und Sasgawa (Sasgawa F and Tajima K (1993) Cablos 9: 147-152).

"Essential properties" of the Tocen-1 protein described by SEQ ID NO: 2 means in particular the property of increasing the vitamin E content in the case of transgenic expression in a plant organism in comparison to an identical but not transgenic plant organism according to the definition given above. In a preferred embodiment, at least one property selected from the following group also applies as essential properties:

• a) a molecular weight in a range from approximately 10 to approximately 25 kDa, preferably approximately 15 to approximately 20 kDa, particularly preferably approximately 17 kDa
• b) a theoretical isoelectric point in a range from approximately 8 to approximately 9.5, preferably approximately 8.4 to approximately 9.1, particularly preferably approximately pH 8.7
• c) an acidic character, preferably with a total net charge at neutral pH in a range from approximately 2 to approximately 3, preferably approximately 2.2 to approximately 2.8, particularly preferably approximately 2.6,
• d) a helical secondary structure according to computer-aided prediction, for example with the program for secondary structure prediction in GenomMax v.3.1 (InforMax, Inc.) based on the work of Qian (Qian N and Sejnowski T (1988) J Mol Biol 202: 865-884) and Sasgawa (Sasgawa F and Tajima K (1993) Cablos 9: 147-152).

Functional equivalents from other organisms, for example, their genomic sequence from plant organisms is known in whole or in part, such as from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum or Solanum tuberosum - can e.g. B. by database search in Sequence databases such as GenBank or screening of gene or cDNA banks - z. B. using the sequence of SEQ ID NO: 1 or one some of them as a search sequence or probe - can be found. Mutations include substitutions, additions, deletions, Inversion or insertions of one or more amino acid residues.

Said functional equivalents preferably have homology of at least 50%, particularly preferably at least 65%, particularly preferred at least 80%, most preferred at least 90% of the protein with SEQ ID NO: 2 homology over at least 30 amino acids is preferred at least 60 amino acids, particularly preferably at least 90 Amino acids, most preferred over the entire length of the Tocen-1 Polypeptides according to SEQ ID NO: 2.

Homology between two polypeptides means the identity of the amino acid sequence over the respective sequence length, which is calculated by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following parameters becomes:
Gap Weight: 8, Length Weight: 2
Average Match: 2,912, Average Mismatch: -2,003

An example is a sequence that has a homology of at least 80% protein based with the sequence SEQ ID NO: 2 has understood a sequence that when compared with the sequence SEQ ID NO: 2 according to the above program algorithm The above parameter set has a homology of at least 80%.

Functional equivalents also include those proteins that encoded by nucleic acid sequences that have homology of at least 50%, particularly preferably at least 65%, particularly preferred at least 80%, most preferred at least 90% of the nucleic acid sequence with SEQ ID NO: 1 to have. The homology extends over at least 100 bases, preferably at least 200 bases particularly preferred at least 300 bases, most preferred over the total Length of the sequence according to SEQ ID NO: 1.

Homology between two nucleic acid sequences is understood to mean the identity of the two nucleic acid sequences over the respective sequence length, which by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25: 3389ff) using the following parameters:
Gap Weight: 50 Length Weight: 3
Average Match: 10 Average Mismatch: 0

An example is a sequence that has a homology of at least 80% nucleic acid based on the sequence SEQ ID NO: 1, understood a sequence that occurs in a Comparison with the sequence SEQ ID NO: 1 according to the above Program algorithm with the above parameter set a homology of has at least 80%.

Functional equivalents also include those proteins that can be encoded by nucleic acid sequences which under Standard conditions with one of those described by SEQ ID NO: 1 Nucleic acid sequence that is complementary to this Hybridize nucleic acid sequence or parts of the aforementioned and the essential properties of the protein described by SEQ ID NO: 2.

"Standard hybridization conditions" is to be understood broadly and means stringent as well as less stringent Hybridization conditions. Such hybridization conditions are among others at Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) 6.3.1-6.3.6. described.

For example, the conditions during the washing step be selected from the range of conditions limited by low stringency (approximately 2X SSC at 50 ° C) and those with high stringency (with approximately 0.2X SSC at 50 ° C preferably at 65 ° C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). In addition, the temperature during the washing step of low stringent conditions at room temperature, around 22 ° C, raised to more stringent conditions at around 65 ° C become. Both parameters, salt concentration and temperature, can can be varied simultaneously, also one of the two Parameters kept constant and only the other can be varied.

• 1. Hybridisierungbedingungen zum Beispiel aus nachfolgenden Bedingungen ausgewählt sein:
  • a) 4X SSC bei 65°C,
  • b) 6X SSC bei 45°C,
  • c) 6X SSC, 100 µg/ml denaturierter, fragmentierte Fischsperma-DNA bei 68°C,
  • d) 50% Formamid, 4X SSC bei 42°C,
  • e) 2X oder 4X SSC bei 50°C (schwach stringente Bedingung),
  • f) 30 bis 40% Formamid, 2X oder 4X SSC bei 42°C (schwach stringente Bedingung).
• 2. Waschschritte können zum Beispiel aus nachfolgenden Bedingungen ausgewählt sein:
  • a) 0,015 M NaCl/0,0015 M Natriumcitrat/0,1% SDS bei 50°C.
  • b) 0,1X SSC bei 65°C.
  • c) 0,1X SSC, 0,5% SDS bei 68°C.
  • d) 0,1X SSC, 0,5% SDS, 50% Formamid bei 42°C.
  • e) 0,2X SSC, 0,1% SDS bei 42°C.
  • f) 2X SSC bei 65°C (schwach stringente Bedingung).

Denaturing agents such as formamide or SDS can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C. Some exemplary conditions for hybridization and washing step are given as a result:

• 1. Hybridization conditions can be selected, for example, from the following conditions:
  • a) 4X SSC at 65 ° C,
  • b) 6X SSC at 45 ° C,
  • c) 6X SSC, 100 µg / ml denatured, fragmented fish sperm DNA at 68 ° C,
  • d) 50% formamide, 4X SSC at 42 ° C,
  • e) 2X or 4X SSC at 50 ° C (weakly stringent condition),
  • f) 30 to 40% formamide, 2X or 4X SSC at 42 ° C (weakly stringent condition).
• 2. Washing steps can be selected, for example, from the following conditions:
  • a) 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% SDS at 50 ° C.
  • b) 0.1X SSC at 65 ° C.
  • c) 0.1X SSC, 0.5% SDS at 68 ° C.
  • d) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C.
  • e) 0.2X SSC, 0.1% SDS at 42 ° C.
  • f) 2X SSC at 65 ° C (weakly stringent condition).

Another object of the invention relates to transgenic Expression constructs that show transgenic expression of the protein encoded by SEQ ID NO: 2 or a functional equivalent the same or a functionally equivalent part of the aforementioned in a plant organism or a tissue, Organ, part or cell of said plant organism can guarantee.

In said transgenic expression constructs is a Nucleic acid molecule coding for a protein described by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the aforementioned preferably in functional combination with at least one genetic control element (e.g. a promoter) that a transgenic expression in a plant organism or a tissue, organ, part or cell of the same.

A functional link is understood to mean Example the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example the sequence according to SEQ ID NO: 1) and possibly other regulatory elements such as a terminator such that each of the regulative elements its function in transgenic expression of the Nucleic acid sequence can meet. This is not necessarily one direct linkage in the chemical sense is required. genetic Control sequences, such as enhancer sequences, can their function also from more distant positions or even from other DNA molecules on the target sequence. Arrangements are preferred in which the transgene is to be expressed Nucleic acid sequence behind the sequence that acts as a promoter is positioned so that both sequences covalently with each other are connected. The distance between the Promoter sequence and the transgene to be expressed Nucleic acid sequence less than 200 base pairs, particularly preferred smaller than 100 base pairs, very particularly preferably smaller than 50 base pairs.

Establishing a functional link as well Production of a transgenic expression construct can be carried out using common recombination and cloning techniques realized as in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and at Gelvin et al. (1990) In: Plant Molecular Biology Manual are. However, further sequences can also be between the two sequences positioned, for example, the function of a linker with certain restriction enzyme interfaces or one Have signal peptides. The insertion of sequences for Expression of fusion proteins result. This can preferably transgenic expression construct, consisting of a linkage of Promoter and nucleic acid sequence to be expressed, integrated exist in a vector and through, for example, transformation can be inserted into a plant genome.

However, there are also those under a transgenic expression construct Understand constructions in which the nucleic acid sequence coding for the protein encoded by SEQ ID NO: 2 or one functional equivalent of the same or a functional equivalent part of the above - for example, by a homologous Recombination - so behind an endogenous plant promoter is placed that the transgenic expression of said Ensured nucleic acid sequence.

Plant-specific promoters basically mean everyone Promoter that expresses genes, in particular Foreign genes, in plants or plant parts, cells, tissues, - can control cultures. The expression for example, constitutive, inducible or developmental.

• a) Konstitutive Promotoren
  "Konstitutive" Promotoren meint solche Promotoren, die eine Expression in zahlreichen, bevorzugt allen, Geweben über einen größeren Zeitraum der Pflanzenentwicklung, bevorzugt zu allen Zeitpunkten der Pflanzenentwicklung, gewährleisten (Benfey et al. (1989) EMBO J 8: 2195-2202). Vorzugsweise verwendet man insbesondere einen pflanzlichen Promotor oder einen Promotor, der einem Pflanzenvirus entstammt. Insbesondere bevorzugt ist der Promotor des 35S-Transkriptes des CaMV Blumenkohlmosaikvirus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) oder der 19S CaMV Promotor (US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8: 2195-2202). Ein weiterer geeigneter konstitutiver Promotor ist der "Rubisco small subunit (SSU)"-Promotor (US 4,962,028), der LeguminB-Promotor (GenBank Acc.-Nr. X03677), der Promotor der Nopalinsynthase aus Agrobacterium, der TR-Doppelpromotor, der OCS (Octopin Synthase) Promotor aus Agrobacterium, der Ubiquitin Promotor (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), den Ubiquitin 1 Promotor (Christensen et al. (1992) Plant Mol Biol 18: 675-689; Bruce et al. (1989) Proc Natl Acad Sci USA 86: 9692-9696), den Smas Promotor, den Cinnamylalkoholdehydrogenase-Promotor (US 5,683,439), die Promotoren der vakuolärer ATPase Untereinheiten oder der Promotor eines prolinreichen Proteins aus Weizen (WO 91/13991), sowie weitere Promotoren von Genen, deren konstitutive Expression in Pflanzen dem Fachmann bekannt ist.
• b) Gewebespezifische Promotoren
  Bevorzugt sind ferner Promotoren mit Spezifitäten für die Antheren, Ovarien, Blüten, Blätter, Stengel, Wurzeln und Samen.
  Samenspezifische Promotoren wie zum Beispiel der Promotor des Phaseolins (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1(9): 839-53), des 25 Albumingens (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), des Legumins (Shirsat et al. (1989) Mol Gen Genet 215(2): 326-331), des USP (unknown seed protein; Baumlein H et al. (1991) Mol Gen Genet 225(3): 459-67), des Napin Gens (US 5,608,152; Stalberg K et al. (1996) L Planta 199: 515-519), des Saccharosebindeproteins (WO 00/26388) oder der Legumin B4-Promotor (LeB4; Baumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baumlein H et al. (1992) Plant J 2(2): 233-9; Fiedler U et al. (1995) Biotechnology (NY) 13(10): 1090f), der Oleosin-Promoter aus Arabidopsis (WO 98/45461), der Bce4-Promoter aus Brassica (WO 91/13980). Weitere geeignete samenspezifische Promotoren sind die der Gene kodierend für das "High Molecular Weight Glutenin" (HMWG), Gliadin, Verzweigungsenzym, ADP Glucose Pyrophosphatase (AGPase) oder die Stärkesynthase. Bevorzugt sind ferner Promotoren, die eine samenspezifische Expression in Monokotyledonen wie Mais, Gerste, Weizen, Roggen, Reis etc. erlauben. Vorteilhaft eingesetzt werden können der Promoter des lpt2 oder lpt1-Gen (WO 95/15389, WO 95/23230) oder die Promotoren beschrieben in WO 99/16890 (Promotoren des Hordein-Gens, des Glutelin-Gens, des Oryzin-Gens, des Prolamin-Gens, des Gliadin-Gens, des Glutelin-Gens, des Zein- Gens, des Kasirin-Gens oder des Secalin-Gens). Weitere samenspezifische Promotoren sind beschrieben in WO 89/03887.
  Knollen-, Speicherwurzel- oder Wurzel-spezifische Promotoren wie beispielsweise der Patatin Promotor Klasse I (B33), der Promotor des Cathepsin D Inhibitors aus Kartoffel.
  Blattspezifische Promotoren wie Promotor der cytosolischen FBPase aus Kartoffel (WO 97/05900), der SSU Promotor (small subunit) der Rubisco (Ribulose-1,5-bisphosphatcarboxylase) oder der ST-LSI Promotor aus Kartoffel (Stockhaus et al. (1989) EMBO J 8: 2445-2451).
  Blütenspezifische Promotoren wie beispielsweise der Phytoen Synthase Promotor (WO 92/16635) oder der Promotor des P-rr Gens (WO 98/22593).
  Antheren-spezifische Promotoren wie den 5126-Promotor (US 5,689,049, US 5,689,051), den glob-1 Promotor und den γ-Zein Promotor.
• c) Chemisch induzierbare Promotoren
  Die transgenen Expressionskonstrukte können auch einen chemisch induzierbaren Promotor enthalten (Übersichtsartikel: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), durch den die Expression des exogenen Gens in der Pflanze zu einem bestimmten Zeitpunkt gesteuert werden kann. Derartige Promotoren, wie z. B. der PRP1 Promotor (Ward et al. (1993) Plant Mol Biol 22: 361-366), durch Salicylsäure induzierbarer Promotor (WO 95/19443), ein durch Benzolsulfonamid-induzierbarer Promotor (EP 0 388 186), ein durch Tetrazyklin-induzierbarer Promotor (Gatz et al. (1992) Plant J 2: 397-404), ein durch Abscisinsäure induzierbarer Promotor (EP 0 335 528) bzw. ein durch Ethanol- oder Cyclohexanoninduzierbarer Promotor (WO 93/21334) können ebenfalls verwendet werden.
• d) Stress- oder Pathogen-induzierbare Promotoren
  Ferner sind Promotoren bevorzugt, die durch biotischen oder ablotischen Stress induziert werden wie beispielsweise der pathogen-induzierbare Promotor des PRP1-Gens (Ward et al. (1993) Plant Mol Biol 22: 361-366), der hitzeinduzierbare hsp70- oder hsp80-Promoter aus Tomate (US 5,187,267), der kälteinduzierare alpha-Amylase Promoter aus der Kartoffel (WO 96/12814), der licht-induzierbare PPDK Promotor oder der verwundungsinduzierte pinII-Promoter (EP 375091).
  Pathogen-induzierbare Promotoren umfassen die von Genen, die infolge eines Pathogenbefalls induziert werden wie beispielweise Gene von PR-Proteinen, SAR-Proteinen, β-1,3-Glucanase, Chitinase usw. (beispielsweise Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes, et al. (1992) The Plant Cell 4: 645-656; Van Loon (1985) Plant Mol Viral 4: 111-116; Marineau et al. (1987) Plant Mol Biol 9: 335-342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2: 325-342; Somssich et al. (1986) Proc Natl Acad Sci USA 83: 2427-2430; Somssich et al. (1988) Mol Gen Genetics 2: 93-98; Chen et al. (1996) Plant J 10: 955-966; Zhang and Sing (1994) Proc Natl Acad Sci USA 91: 2507-2511; Warner, et al. (1993) Plant J 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968(1989).
  Umfasst sind auch verwundungs-induzierbare Promotoren wie der des pinII Gens (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), des wun1 und wun2-Gens (US 5,428,148), des win1- und win2-Gens (Stanford et al. (1989) Mol Gen Genet 215: 200-208), des Systemin (McGurl et al. (1992) Science 225: 1570-1573), des WIP1-Gens (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76), des MPI-Gens (Corderok et al. (1994) The Plant J 6(2): 141-150) und dergleichen.
• e) Entwicklungsabhängige Promotoren
  Weitere geeignete Promotoren sind beispielsweise fruchtreifung-spezifische Promotoren, wie beispielsweise der fruchtreifung-spezifische Promotor aus Tomate (WO 94/21794, EP 409 625). Entwicklungsabhängige Promotoren schließt zum Teil die Gewebespezifischen Promotoren ein, da die Ausbildung einzelner Gewebe naturgemäß entwicklungsabhängig erfolgt.

Preferred are:

• a) Constitutive promoters
  “Constitutive” promoters mean those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development (Benfey et al. (1989) EMBO J 8: 2195-2202). In particular, a plant promoter or a plant virus-derived promoter is preferably used. Particularly preferred is the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140 : 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) or the 19S CaMV promoter (US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8: 2195-2202) , Another suitable constitutive promoter is the "Rubisco small subunit (SSU)" promoter (US 4,962,028), the LeguminB promoter (GenBank Acc. No. X03677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (Octopin Synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), the Ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18: 675-689 ; Bruce et al. (1989) Proc Natl Acad Sci USA 86: 9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (US 5,683,439), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91 / 13991), as well as further promoters of genes whose constitutive expression in plants is known to the person skilled in the art.
• b) Tissue-specific promoters
  Also preferred are promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds.
  Seed-specific promoters such as, for example, the promoter of phaseoline (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53), of 25 albuming gene (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), legumin (Shirsat et al. (1989) Mol Gen Genet 215 (2): 326-331), USP (unknown seed protein; Baumlein H et al. (1991) Mol Gen Genet 225 (3) : 459-67), the Napin gene (US 5,608,152; Stalberg K et al. (1996) L Planta 199: 515-519), the sucrose binding protein (WO 00/26388) or the legumin B4 promoter (LeB4; Baumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baumlein H et al. (1992) Plant J 2 (2): 233-9; Fiedler U et al. (1995) Biotechnology (NY) 13 (10): 1090f), the oleosin promoter from Arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980). Further suitable seed-specific promoters are those of the genes coding for "high molecular weight glutenin" (HMWG), gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch synthase. Also preferred are promoters that allow seed-specific expression in monocots such as corn, barley, wheat, rye, rice etc. The promoter of the lpt2 or lpt1 gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzine gene, etc.) can be used advantageously Prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the kasirin gene or the secalin gene). Further seed-specific promoters are described in WO 89/03887.
  Tuber-, storage root- or root-specific promoters such as the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato.
  Leaf-specific promoters such as a promoter of the cytosolic FBPase from potatoes (WO 97/05900), the SSU promoter (small subunit), the Rubisco (ribulose-1,5-bisphosphate carboxylase) or the ST-LSI promoter from potatoes (Stockhaus et al. (1989) EMBO J 8: 2445-2451).
  Flower-specific promoters such as the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
  Anther-specific promoters such as the 5126 promoter (US 5,689,049, US 5,689,051), the glob-1 promoter and the γ-zein promoter.
• c) Chemically inducible promoters
  The transgenic expression constructs can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a specific point in time can. Such promoters, such as. B. the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracycline inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), a promoter inducible by abscisic acid (EP 0 335 528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334) can also be used ,
• d) stress or pathogen inducible promoters
  Also preferred are promoters that are induced by biotic or ablotic stress, such as the pathogen-inducible promoter of the PRP1 gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (US 5,187,267), the cold-inducing alpha-amylase promoter from the potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinII promoter (EP 375091).
  Pathogen-inducible promoters include those of genes induced by pathogen attack such as genes from PR proteins, SAR proteins, β-1,3-glucanase, chitinase etc. (e.g. Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes, et al. (1992) The Plant Cell 4: 645-656; Van Loon (1985) Plant Mol Viral 4: 111-116; Marineau et al. (1987) Plant Mol Biol 9: 335-342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2: 325-342; Somssich et al. (1986) Proc Natl Acad Sci USA 83: 2427-2430; Somssich et al. (1988) Mol Gen Genetics 2: 93-98; Chen et al. (1996) Plant J 10: 955-966; Zhang and Sing (1994) Proc Natl Acad Sci USA 91: 2507-2511; Warner, et al. (1993) Plant J 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968 (1989).
  Also included are wound-inducible promoters such as that of the pinII gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wun1 and wun2 genes (US 5,428,148), the win1 and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the Systemin (McGurl et al. (1992) Science 225: 1570-1573), the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76), the MPI gene (Corderok et al. (1994) The Plant J 6 ( 2): 141-150) and the like.
• e) Development-dependent promoters
  Further suitable promoters are, for example, fruit-ripening-specific promoters, such as, for example, the fruit-ripening-specific promoter from tomato (WO 94/21794, EP 409 625). Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.

Constitutive, seed-specific as well as leaf-specific promoters.

Further promoters can also functionally function with the expressing nucleic acid sequence can be linked that a transgenic expression in other plant tissues or in others Enable organisms such as E.coli bacteria. As Plant promoters come in principle all described above Promoters in question.

The in the transgenic expression constructs according to the invention or contain transgenic expression vectors Nucleic acid sequences can be linked to other genetic control sequences be functionally linked next to a promoter. The concept of genetic control sequences is to be understood broadly and means all those sequences that have an impact on the formation or the function of a transgenic according to the invention Have expression construct. Modify genetic control sequences to Example the transcription and translation in prokaryotic or eukaryotic organisms. Preferably, the transgenic expression constructs according to the invention 5 'upstream of the respective transgene to be expressed Nucleic acid sequence a plant specific promoter and 3'-downstream a terminator sequence as an additional genetic Control sequence, as well as any other usual regulatory Elements, each functionally linked to the transgene nucleic acid sequence to be expressed.

Genetic control sequences also include other promoters Promoter elements or minimal promoters that the can modify expression-controlling properties. So through genetic control sequences, for example the tissue-specific ones Expression also depends on certain stress factors respectively. Corresponding elements are for example for Water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131-17135) and heat stress (Schoffl F et al. (1989) Mol Gen Genetics 217 (2-3): 246-53).

Further advantageous control sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast or Fungus promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters can with their Regulatory sequences like the above for the inventive methods are used. You can also synthetic promoters can be used advantageously.

Genetic control sequences also include the 5 'untranslated regions, introns or 3' non-coding region of Genes such as the Actin-1 intron, or the Adh1-S Introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It is been shown to have significant functions in can play a role in regulating gene expression. So it was demonstrated that 5'-untranslated sequences made the transient Can enhance expression of heterologous genes. Exemplary for Translation enhancer is the 5 'leader sequence from the tobacco To name the mosaic virus (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. You can also use the Promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).

The transgenic expression construct can advantageously be a or several so-called "enhancer sequences" functionally linked to the promoter containing an increased transgenic Enable expression of the nucleic acid sequence. Also at the 3 'end of the transgenic nucleic acid sequences to be expressed additional advantageous sequences are inserted, such as further regulatory elements or terminators. The Nucleic acid sequences to be expressed transgenically can be in a or several copies can be contained in the gene construct.

Suitable polyadenylation signals as control sequences vegetable polyadenylation signals, preferably those which essentially T-DNA polyadenylation signals from Agrobacterium tumefaciens, especially gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J 3: 835 ff) or functional equivalents thereof. examples for particularly suitable terminator sequences are the OCS (octopine Synthase) terminator and the NOS (nopaline synthase) terminator.

Control sequences are also to be understood as those which a homologous recombination or insertion into the genome of a Allow host organism or removal from the genome allow. With homologous recombination, for example coding sequence of a specific endogenous gene against the can be exchanged specifically for a sequence coding for dsRNA. Methods like cre / lox technology allow one tissue-specific, possibly inducible removal of the transgenic expression construct from the genome of the host organism (Sauer B (1998) Methods 14 (4): 381-92). Here are certain flanking sequences added to the target gene (lox sequences), the later allow removal using the cre recombinase.

• a) Selektionsmarker, die eine Resistenz gegen einen Metabolismusinhibitor wie 2-Desoxyglucose-6-phosphat (WO 98/45456), Antibiotika oder Biozide, bevorzugt Herbizide, wie zum Beispiel Kanamycin, G 418, Bleomycin, Hygromycin, oder Phosphinotricin etc. verleihen. Besonders bevorzugte Selektionsmarker sind solche die eine Resistenz gegen Herbizide verleihen. Beispielhaft seien genannt: DNA Sequenzen, die für Phosphinothricinacetyltransferasen (PAT) kodieren und Glutaminsynthaseinhibitoren inaktivieren (bar und pat Gen), 5-Enolpyruvylshikimat-3-phosphatsynthasegene (EPSP Synthasegene), die eine Resistenz gegen Glyphosat® (N-(phosphonomethyl)glycin) verleihen, das für das Glyphosat® degradierende Enzyme kodierende gox Gen (Glyphosatoxidoreduktase), das deh Gen (kodierend für eine Dehalogenase, die Dalapon inaktiviert), Sulfonylurea- und Imidazolinon inaktivierende Acetolactatsynthasen sowie bxn Gene, die für Bromoxynil degradierende Nitrilaseenzyme kodieren, das aasa-Gen, das eine Resistenz gegen das Antibiotikum Apectinomycin verleih, das Streptomycinphosphotransferase (SPT) Gen, das eine Resistenz gegen Streptomycin gewährt, das Neomycinphosphotransferas (NPTII) Gen, das eine Resistenz gegen Kanamycin oder Geneticidin verleiht, das Hygromycinphosphotransferase (HPT) Gen, das eine Resistenz gegen Hygromycin vermittelt, das Acetolactatsynthas Gen (ALS), das eine Resistenz gegen Sulfonylharnstoff-Herbizide verleiht (z. B. mutierte ALS- Varianten mit z. B. der S4 und/oder Hra Mutation).
• b) Reportergene, die für leicht quantifizierbare Proteine kodieren und über Eigenfarbe oder Enzymaktivität eine Bewertung der Transformationseffizienz oder des Expressionsortes oder -zeitpunktes gewährleisten. Ganz besonders bevorzugt sind dabei Reporter-Proteine (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1): 29-44) wie das "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8(5): 777-784), die Chloramphenicoltransferase, eine Luziferase (Ow et al. (1986) Science 234: 856-859), das Aequorin- Gen (Prasher et al. (1985) Biochem Biophys Res Commun 126(3): 1259-1268), die β-Galactosidase, ganz besonders bevorzugt ist die β-Glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).
• c) Replikationsursprünge, die eine Vermehrung der erfindungsgemäßen transgenen Expressionskonstrukte oder transgenen Expressionsvektoren in zum Beispiel E.coli gewährleisten. Beispielhaft seien genannt ORI (origin of DNA replication), der pBR322 ori oder der P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2^nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• d) Elemente, die für eine Agrobakterium vermittelte Pflanzentransformation erforderlich sind, wie zum Beispiel die rechte oder linke Begrenzung der T-DNA oder die vir-Region.

A transgenic expression construct and / or the transgenic expression vectors derived from it can contain further functional elements. The term functional element is to be understood broadly and means all those elements which have an influence on the production, multiplication or function of the transgenic expression constructs according to the invention, the transgenic expression vectors or the transgenic organisms. Examples include, but are not limited to:

• a) Selection markers which confer resistance to a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc. Particularly preferred selection markers are those which confer resistance to herbicides. Examples include: DNA sequences which code for phosphinothricin acetyltransferases (PAT) and inactivate glutamine synthase inhibitors (bar and pat gene), 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which have resistance to Glyphosat® (Ncin) phosphonomethyl confer the gox gene (glyphosate oxidoreductase) coding for the glyphosate® degrading enzyme, the deh gene (coding for a dehalogenase which inactivates dalapon), sulfonylurea and imidazolinone inactivating acetolactate synthases, and bxn genes which degenerate nitrodenase-degrading enzyme, bromoxynilase Gene conferring resistance to the antibiotic apectinomycin, the streptomycin phosphotransferase (SPT) gene conferring resistance to streptomycin, the neomycin phosphotransferas (NPTII) gene conferring resistance to kanamycin or geneticidin, the hygromycin phosphotransferase (HPT) gene, the Resistance to hygromycin mediates the Acetolacta tsynthas gene (ALS), which confers resistance to sulfonylurea herbicides (e.g. B. mutated ALS variants with z. B. the S4 and / or Hra mutation).
• b) reporter genes which code for easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an assessment of the transformation efficiency or the place or time of expression. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as the "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784), chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234: 856-859), the aequorin gene (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3) : 1259-1268), the β-galactosidase, the β-glucuronidase is very particularly preferred (Jefferson et al. (1987) EMBO J 6: 3901-3907).
• c) origins of replication, which ensure an increase in the transgenic expression constructs or transgenic expression vectors according to the invention in, for example, E. coli. Examples are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al .: Molecular Cloning. A Laboratory Manual, ^2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• d) Elements which are required for an agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.

For the selection of successfully homologously recombined or transformed cells, it is usually necessary to use one selectable marker to introduce the successful recombined cells resistance to a biocide (for Example a herbicide), a metabolism inhibitor such as 2-Deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic. The selection marker allows the selection of the transformed ones Untransformed cells (McCormick et al. (1986) Plant Cell Reports 5: 81-84).

In addition, said transgenic expression constructs or contain transgenic expression vectors nucleic acid sequences, which is not encoded for the protein by SEQ ID NO: 2 functional equivalent of the same or a functional encode equivalent part of the above, and their transgenes Expression for an additional boost in vitamin E-biosynthesis leads (as a result of pro-VitE). This additionally transgenic The expressed pro-VitE nucleic acid sequence can - but is exemplary non-limiting - selected from coding from nucleic acids for homogentisate phytyltransferase, 2-methyl-6-plastoquinone methyl transferase, tocopherol cyclase, γ-tocopherol methyl transferase, Hydroxyphenylpyruvate dioxygenase, tyrosine aminotransferase, tyrA, Geranylgeranylpyrophosphatreduktase. With the aforementioned the desired effect is guaranteed by overexpression. However, a corresponding effect can also be achieved by expressing a a pro-VitE nucleic acid sequence can be achieved z. B. as antisense RNA or double-stranded RNA, the suppression of Expression of certain genes causes. Suitable target genes would be here exemplary but not restrictive - the Homogentisate. Corresponding sequences are known to the person skilled in the art and from databases or corresponding cDNA banks of the respective Plants easily accessible. The skilled worker is aware that also several different of the above additional ones Expressions of pro-VitE nucleic acid sequences in the context of Invention can be combined.

The introduction of a transgenic according to the invention Expression construct in an organism or cells, tissues, organs, Parts or seeds of the same (preferably in plants or plant cells, tissues, organs, parts or seeds), can be beneficial can be realized using vectors in which the transgenic expression constructs are included. Vectors can exemplary plasmids, cosmids, phages, viruses or Agrobacteria. The transgenic expression construct can be found in the vector (preferably a plasmid vector) via a suitable one Restriction interface will be introduced. The resulting one transgenic expression vector is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the combined vector using methods familiar to the person skilled in the art won. Restriction analysis and sequencing can do this serve to check the cloning step. Are preferred such vectors that provide stable integration of the transgenic Enable expression construct in the host genome.

The production of a transformed organism (or a transformed cell or tissue) requires that the corresponding DNA (e.g. the expression vector), RNA or protein is introduced into the corresponding host cell. For this Process that takes the form of a transformation (or transduction or Transfection) is a variety of methods available (Keown et al. (1990) Methods in Enzymology 185: 527-537). For example, the DNA or RNA can go straight through Microinjection or by bombardment with DNA-coated Microparticles are introduced. The cell can also be chemically for example with polyethylene glycol, can be permeabilized, so that the DNA can get into the cell by diffusion. The DNA can also be obtained by protoplast fusion with others DNA-containing units such as minicells, cells, or lysosomes Liposomes are done. Electroporation is another suitable one Method of introducing DNA in which the cells are reversible be permeabilized by an electrical pulse. Appropriate methods are described (for example at Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol Gen Genet 228: 104-112; Guerche et al. (1987) Plant Science 52: 111-116; Neuhause et al. (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature 327: 70-73; Howell et al. (1980) Science 208: 1265; Horsch et al. (1985) Science 227: 1229-1231; DeBlock et al. (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).

In plants, the methods described are used Transformation and regeneration from plants Plant tissues or plant cells for transient or stable Transformation used. Suitable methods are, above all Protoplast transformation induced by polyethylene glycol DNA uptake, the biolistic method with the gene gun, the so-called "particle bombardment" method, electroporation, the incubation of dry embryos in DNA-containing solution and the microinjection.

In addition to these "direct" transformation techniques, a Transformation also by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes can be performed. The Agrobacterium -mediated transformation is best for dicotyledonous plant cells suitable. The procedures are described for example by Horsch RB et al. (1985) Science 225: 1229f).

If Agrobacteria are used, this is transgenic Integrate expression construct into special plasmids, either in an intermediate vector (English: shuttle or intermediate vector) or a binary vector. If a Ti or Ri plasmid is used Transformation to be used is at least the right one Boundary, but mostly the right and the left boundary the Ti or Ri plasmid T-DNA as a flanking region with the connected to be introduced transgenic expression construct.

Binary vectors are preferably used. Binary vectors can replicate in both E.coli and Agrobacterium. They usually contain a selection marker gene and one Left or polylinker flanked by the right and left T-DNA restriction sequence. You can go straight to Agrobacterium can be transformed (Holsters et al. (1978) Mol Gen Genet 163: 181-187). The selection marker gene allows selection transformed agrobacteria and is for example the nptII gene, which confers resistance to kanamycin. In this case Agrobacterium acting as the host organism should already be a Contain plasmid with the vir region. This is for the Transfer of T-DNA to the plant cell required. On Agrobacterium thus transformed can be used for transformation plant cells can be used. The use of T-DNA for Transformation of plant cells has been studied intensively and (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO 4: 277-287). Different binary vectors are known and some are commercially available such as Example pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).

Further promoters suitable for expression in plants are (Rogers et al. (1987) Meth in Enzymol 153: 253-277; Schardl et al. (1987) Gene 61: 1-11; Berger et al. (1989) Proc Natl Acad Sci USA 86: 8402-8406).

Direct transformation techniques are suitable for everyone Organism and cell type. In the case of injection or Electroporation of DNA or RNA into plant cells are not made special demands on the plasmid used. Simple plasmids such as the pUC series can be used. Should complete plants from the transformed cells be regenerated, so it is necessary that on the Plasmid is an additional selectable marker gene.

Stably transformed cells d. H. those that the introduced DNA integrated into the DNA of the host cell can contain from untransformed can be selected if a selectable marker is part of the introduced DNA. As a marker can act as an example of any gene that is resistant to Antibiotics or herbicides (such as kanamycin, G 418, bleomycin, Hygromycin or phosphinotricin etc.) is able to confer (see above). Transformed cells that express such a marker gene are able to in the presence of concentrations of one corresponding antibiotic or herbicide to survive the one kill untransformed wild type. Example are mentioned above and preferably include the bar gene that resistance to the herbicide Phosphinotricin confers (Rathore KS et al. (1993) Plant Mol Biol 21 (5): 871-884), the nptII gene that resistance to kanamycin conferred the hpt gene conferring resistance to hygromycin, or the EPSP gene, which is resistant to the herbicide glyphosate gives. The selection marker allows the selection of transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84). The plants obtained can in are bred and crossed in the usual way. Two or more Generations should be cultivated to ensure that genomic integration is stable and inheritable.

The above methods are described in, for example That B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by SD Kung and R Wu, Academic Press, pp. 128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225). The expression construct to be transgenic is preferably converted into one Vector cloned that is suitable for Agrobacterium tumefaciens transform, for example pBin19 (Bevan et al. (1984) Nucl Acids Res 12: 8711f).

Once a transformed plant cell has been made, a whole plant using the skilled person known methods can be obtained. Here you go as an example from callus cultures. From these still undifferentiated Cell masses can cause sprout and root formation in a known manner be induced. The sprouts obtained can be planted out and be bred.

The person skilled in the art is also aware of processes for extracting from plant cells Regenerate parts of plants and whole plants. For example For this purpose, methods are described by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14: 273-278; Jahne et al. (1994) Theor Appl Genet 89: 525-533 used.

• a) die Nukleinsäuresequenz kodierend für ein Tocen-1 Protein gemäß SEQ ID NO: 2, ein funktionelles Äquivalent desselben oder ein funktionell äquivalentes Teil der vorgenannten, oder
• b) eine mit besagter Nukleinsäuresequenz unter a) funktionell verknüpfte genetische Kontrollsequenz, zum Beispiel ein Promotor, oder
• c) (a) und (b)

sich nicht in ihrer natürlichen, genetischen Umgebung befinden oder durch gentechnische Methoden modifiziert wurden, wobei die Modifikation beispielhaft eine Substitutionen, Additionen, Deletionen, Inversion oder Insertionen eines oder mehrerer Nukleotidreste sein kann. Natürliche genetische Umgebung meint den natürlichen chromosomalen Locus in dem Herkunftsorganismus oder das Vorliegen in einer genomischen Bibliothek. Im Fall einer genomischen Bibliothek ist die natürliche, genetische Umgebung der Nukleinsäuresequenz bevorzugt zumindest noch teilweise erhalten. Die Umgebung flankiert die Nukleinsäuresequenz zumindest an einer Seite und hat eine Sequenzlänge von mindestens 50 bp, bevorzugt mindestens 500 bp, besonders bevorzugt mindestens 1000 bp, ganz besonders bevorzugt mindestens 5000 bp. Eine natürlich vorkommendes Expressionskonstrukt - beispielsweise die natürlich vorkommende Kombination des Promotors eines Gens kodierend für ein Protein gemäß SEQ ID NO: 2 oder ein funktionelles Äquivalent desselben mit seinen entsprechenden kodierenden Sequenzen wird zu einem transgenen Expressionskonstrukt, wenn diese durch nicht-natürliche, synthetische ("künstliche") Verfahren wie beispielsweise einer Mutagenisierung geändert wird. Entsprechende Verfahren sind beschrieben (US 5,565,350; WO 00/15815; siehe auch oben). "Transgene" means - for example with regard to a nucleic acid sequence, an expression construct or an expression vector containing said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression construct or expression vector - all such constructions which have been obtained by genetic engineering methods, in which either

• a) the nucleic acid sequence coding for a Tocen-1 protein according to SEQ ID NO: 2, a functional equivalent thereof or a functionally equivalent part of the aforementioned, or
• b) a genetic control sequence which is functionally linked to said nucleic acid sequence under a), for example a promoter, or
• c) (a) and (b)

are not in their natural, genetic environment or have been modified by genetic engineering methods, the modification being for example a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment means the natural chromosomal locus in the organism of origin or the presence in a genomic library. In the case of a genomic library, the natural, genetic environment of the nucleic acid sequence is preferably at least partially preserved. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, very particularly preferably at least 5000 bp. A naturally occurring expression construct - for example the naturally occurring combination of the promoter of a gene coding for a protein according to SEQ ID NO: 2 or a functional equivalent thereof with its corresponding coding sequences becomes a transgenic expression construct if this is caused by non-natural, synthetic (" artificial ") methods such as mutagenization is changed. Corresponding methods are described (US 5,565,350; WO 00/15815; see also above).

"Transgene" means in relation to an expression ("transgene Expression ") prefers all those using a transgenic Expression construct, transgenic expression vector or transgenic organism - according to the definitions given above - realized expressions.

Preferred host or as transgenic organisms Starting organisms are mainly plant organisms according to the above mentioned definition. Are included within the scope of the invention all genera and species of higher and lower plants of the Plant-rich, especially plants that are for extraction of oils such as rapeseed, sunflower, Sesame, safflower, olive tree, soy, corn, wheat and types of nuts. The mature plants, seeds and sprouts are also included and seedlings, as well as parts derived therefrom, propagation material and cultures, for example cell cultures. Ripe plants means Plants at any stage of development beyond Seedling. Seedling means a young, immature plant in one early development stage.

The production of the transgenic organisms can be done with the above described methods for transformation or transfection be realized by organisms.

Another object of the invention relates to the use the transgenic organisms according to the invention and that of them derived cells, cell cultures, parts - such as with transgenic plant organisms roots, leaves etc. -, and transgenic propagation material such as seeds or fruits Manufacture of food or feed, pharmaceuticals or fine chemicals, especially vitamin E or vitamin E Derivatives such as vitamin E acetate.

sequences

• 1. SEQ ID NO: 1 nucleic acid sequence coding for Tocen-1 protein
• 2. SEQ ID NO: 2 protein sequence coding for Tocen-1 protein
• 3. SEQ ID NO: 3 oligonucleotide primers sll0832-5 '
• 4. SEQ ID NO: 4 oligonucleotide primers sll0832-3 '
• 5. SEQ ID NO: 5 nucleic acid sequence coding for the SalI-PacI-ocs-HindIII sequence fragment
• 6. SEQ ID NO: 6 oligo sequence coding for the sense strand a SwaI linker
• 7. SEQ ID NO: 7 oligo sequence coding for the antisense Strand of a SwaI linker
• 8. SEQ ID NO: 8 nucleic acid sequence coding for the Expression vector pSUN2-1
• 9. SEQ ID NO: 9 nucleic acid sequence coding for the rbcS- transit peptide
• 10. SEQ ID NO: 10 nucleic acid sequence coding for the Expression vector pSUN2-2
• 11. SEQ ID NO: 11 oligonucleotide primer 0832ex1-5 '
• 12. SEQ ID NO: 12 oligonucleotide primer 0832ex1-3 '
• 13. SEQ ID NO: 13 nucleic acid sequence coding for the A. thaliana nitrilase-1 promoter
• 14. SEQ ID NO: 14 nucleic acid sequence coding for the transgenic expression vector pSUN2 / NITP / sll0832 / ocs
• 15. SEQ ID NO: 15 nucleic acid sequence coding for the transgenic expression vector pSUN2 / NITP / rbcS / sll0832 / ocs
• 16. SEQ ID NO: 16 nucleic acid sequence coding for the USP Vicia faba promoter in pUC vector
• 17. SEQ ID NO: 17 oligonucleotide primer sll0832ex2-5 '
• 18. SEQ ID NO: 18 oligonucleotide primer Sll0832ex2-3 '
• 19. SEQ ID NO: 19 nucleic acid sequence coding for the transgenic expression vector pSUN2-USPsll0832-Catt
• 20. SEQ ID NO: 20 nucleic acid sequence encode the rbcS MCS-OCS fragment (rbs: rbs transit peptide; MCS: multiple cloning site; OCS: Termination signal of the octopine synthase gene.
• 21. SEQ ID NO: 21 nucleic acid sequence coding for the transgenic expression vector pSUN2 / USP / rbcS / sll0832 / ocs

pictures

Fig. 1 construct card from pCR-Script / sll10832
"A" represents the DNA fragment coding for Tocen-1 (456 base pairs)

Fig. 2 pCR-Script / sll0832 :: tn903 construct card
Fragment A '(268 base pairs) contains the 5' region of Tocen-1, fragment B the transposon 903 (1286 base pairs) and fragment 'A (194 base pairs) the 3' region of Tocen-1. The kanamycin cassette of the tn903 inserted in pCR-Script / sll0832 in such a way that the transcription of the kanamycin resistance gene of the Tn903 runs in the opposite direction to the transcription of the open reading frame of Tocen-1.

Figure 3 Tocopherol production in Synechocystis / sll0832 :: tn903
Two independent Tocen-1 knockout mutants (1 and 2) showed in comparison to the Synechocystis spec. PCC 6803 wild-type cells (WT) showed a significant decrease in tocopherol and tocotrienol production.

Fig. 4 construct map of pSUN2 / NitP / sll0832 / ocs (SEQ ID No: 14)
Fragment "A" (1894 bp): nitrilase-1 promoter
Fragment "B" (456 bp): open reading frame of Tocen-1
Fragment "C" (219 bp): termination signal of the octopine synthase gene.

Fig. 5 pSUN2 / NitP / rbcS / sll0832 / ocs construct map
Fragment "A" (1894 bp): nitrilase-1 promoter
Fragment "B" (167 bp): fragment coding for the rbcS transit peptide
Fragment "C" (456 bp): open reading frame of Tocen-1
Fragment "D" (219 bp): termination signal of the octopine synthase gene.

Fig. 6 pSUN2 / USP / sll10832 / 3'cat construct card
Fragment "A" (674 bp): promoter of the USP gene from Vicia faba
Fragment "B" (456 bp): open reading frame of Tocen-1
Fragment "C" (229 bp): termination signal of the cathepsin D inhibitor gene.

Fig. 7 construction card of pSUN2 / USP / rbcS / sll0832 / ocs
Fragment "A" (674 bp): promoter of the USP gene from Vicia faba
Fragment "B" (174 bp): fragment coding for the rbcS transit peptide
Fragment C (459 bp): open reading frame of Tocen-1
Fragment D (219 bp): termination signal of the octopine synthase gene.

Examples

All chemicals, unless otherwise stated, are from the Companies Fluka (Buchs), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Restriction enzymes, DNA modifying enzymes and molecular biology kits have been developed by the Companies Amersham-Pharmacia (Freiburg), Biometra (Göttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden), Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom). The used Reagents were made according to the manufacturer's instructions used.

The chemical synthesis of oligonucleotides can, for example, in a known manner, according to the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The performed within the scope of the present invention Cloning steps such as B. restriction splits, Agarose gel electrophoresis, purification of DNA fragments, transfer of Nucleic acids on nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of Bacteria, phage propagation and recombinant sequence analysis DNA is, as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules are sequenced using a Laser fluorescence DNA sequencer from ABI using the method by Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74: 5463-5467).

example 1 General procedures

• a) Anzucht von Arabidopsis Pflanzen
  Die Pflanzen werden entweder auf Murashige-Skoog Medium mit 0,5% Saccharose (Ogas et al. (1997) Science 277: 91-94) oder auf Erde gezogen (Focks & Benning (1998) Plant Physiol 118: 91-101). Um einheitliche Keimungs- und Blühzeiten zu erreichen, werden die Samen nach Ausplattieren bzw. Ausstreuen auf Erde zwei Tage bei 4°C stratifiziert. Nach der Blüte werden die Schoten markiert. Entsprechend der Markierungen werden dann Schoten mit einem Alter von 6 bis 20 Tagen nach der Blüte geerntet.
• b) Anzucht von Synechocystis
  Die Zellen von Synechocystis sp. PCC 6803 werden in BG11-Medium normalerweise autotroph kultiviert. Sie haben einen Durchmesser von 2,3 bis 2,5 µm. Bei den hier geschilderten Untersuchungen wurde der Cyanobakterium Synechocystis sp. PCC 6803 Stamm aus der Sammlung von Prof. Dr. Peter Wolk und Prof. Dr. Lee McIntosh (Plant Research Laboratory, Michigan State University, East Lansing, Michigan, USA) eingesetzt. Dieser ist Glukose-tolerant, d. h. er kann auch heterotroph im Dunkeln, mit nur wenigen Minuten schwacher Blaulicht-Beleuchtung pro Tag, wachsen. Diese Kulturbedingungen wurden von Anderson und McIntosh (Anderson und McIntosh (1991) J Bacteriol 173: 2761-2767) entwickelt und "Light-activated heterotrophic growth" (LAHG) genannt. Damit ist es möglich, diese Cyanobakterien ohne laufende Photosynthese und damit ohne Produktion von Sauerstoff zu kultivieren.
  BG 11 Kulturmedium für Synechocystis
  Stammlösung 100 × BG11:
  NaNO₃ 1,76 M = 149,58 g
  MgSO₄ × 7 H₂O 30,4 mM = 7,49 g
  CaCl₂ × 2 H₂O 24,5 mM = 3,6 g
  Zitronensäure 3,12 mM = 0,6 g
  15 Na EDTA pH 8 0,279 mM = 0,104 g
  Die abgewogenen Substanzen werden in 900 ml H₂O gelöst und mit 100 ml des "Trace metal mix stock" 1000x auf 1000 ml aufgefüllt. Diese gewonnene Lösung dient als Stammlösung.
  Trace metall mix stock 1000x:
  H₃BO₃ 46,3 mM = 2,86 g/l
  MnCl₂ × 4 H₂O 4,15 mM = 1,81 g/l
  ZnSO₄ × 7 H₂O 0,77 mM = 0.222 g/l
  Na₂MoO₄ × 2 H₂O 1,61 mM = 0,39 g/l
  CuSO₄ × 5 H₂O 0,32 mM = 0,079 g/l
  Co(NO₃)₂ × 6 H₂O 0,17 mM = 0,0494 g/l
  Für 1 Liter BG11 Kulturlösung werden folgende Lösungen benötigt:
  • 1. 10 ml der Stammlösung 100 × BG 11
  • 2. 1 ml Na₂CO₃ (189 mM)
  • 3. 5 ml TES (1 M, pH 8)
  • 4. 1 ml K₂PO₄ (175 mM)

The Arabidopsis thaliana plant represents a member of the higher plants (seed plants). This plant is closely related to other plant species from the cruciferous family such as B. Brassica napus, but also with other plant families of the dicotyledons. Due to the high degree of homology of their DNA sequences or polypeptide sequences, Arabidopsis thaliana can be used as a model plant for other plant species.

• a) Cultivation of Arabidopsis plants
  The plants are grown either on Murashige-Skoog medium with 0.5% sucrose (Ogas et al. (1997) Science 277: 91-94) or on earth (Focks & Benning (1998) Plant Physiol 118: 91-101). In order to achieve uniform germination and flowering times, the seeds are stratified at 4 ° C for two days after plating or scattering on earth. After flowering, the pods are marked. According to the markings, pods are harvested 6 to 20 days after flowering.
• b) Cultivation of Synechocystis
  The cells of Synechocystis sp. PCC 6803 are usually grown autotrophically in BG11 medium. They have a diameter of 2.3 to 2.5 µm. In the investigations described here, the cyanobacterium Synechocystis sp. PCC 6803 strain from the collection of Prof. Dr. Peter Wolk and Prof. Dr. Lee McIntosh (Plant Research Laboratory, Michigan State University, East Lansing, Michigan, USA). This is glucose-tolerant, which means that it can also grow heterotrophically in the dark with only a few minutes of weak blue light lighting per day. These culture conditions were developed by Anderson and McIntosh (Anderson and McIntosh (1991) J Bacteriol 173: 2761-2767) and called "Light-activated heterotrophic growth" (LAHG). This makes it possible to cultivate these cyanobacteria without ongoing photosynthesis and thus without the production of oxygen.
  BG 11 culture medium for Synechocystis
  Stock solution 100 × BG11:
  NaNO ₃ 1.76 M = 149.58 g
  MgSO ₄ x 7 H ₂ O 30.4 mM = 7.49 g
  CaCl ₂ x 2 H ₂ O 24.5 mM = 3.6 g
  Citric acid 3.12 mM = 0.6 g
  15 Na EDTA pH 8 0.279 mM = 0.104 g
  The weighed substances are dissolved in 900 ml of H ₂ O and made up to 1000 ml with 100 ml of the "Trace metal mix stock" 1000 times. This obtained solution serves as a stock solution.
  Trace metal mix stock 1000x:
  H ₃ BO ₃ 46.3 mM = 2.86 g / l
  MnCl ₂ x 4 H ₂ O 4.15 mM = 1.81 g / l
  ZnSO ₄ x 7 H ₂ O 0.77 mM = 0.222 g / l
  Na ₂ MoO ₄ × 2 H ₂ O 1.61 mM = 0.39 g / l
  CuSO ₄ x 5 H ₂ O 0.32 mM = 0.079 g / l
  Co (NO ₃ ) ₂ x 6 H ₂ O 0.17 mM = 0.0494 g / l
  The following solutions are required for 1 liter of BG11 culture solution:
  • 1. 10 ml of the stock solution 100 × BG 11
  • 2. 1 ml Na ₂ CO ₃ (189 mM)
  • 3.5 ml TES (1 M, pH 8)
  • 4.1 ml K ₂ PO ₄ (175 mM)

While solution 2 and 3 should be sterile filtered, solution 4 must be autoclaved. The entire BG11 Culture solution must be autoclaved before use and then with 1 ml of iron ammonium citrate (6 mg / ml), which was previously sterile was filtered, are added. The iron ammonium citrate should never be autoclaved. For agar plates 1.5% (w / v) Bactoagar are added per liter of BG11 medium.

Example 2 Amplification and cloning from Tocen-1 Synechocystis spec. PCC 6803

The DNA coding for Tocen-1 was determined using the "Polymerase Chain Reaction "(PCR) from Synechocystis spec. PCC 6803 according to the Method according to Crispin A. Howitt (Howitt CA (1996) BioTechniques 21: 32-34) using a sense specific primer (sll0832-5 '; SEQ ID NO: 3) and an antisense specific Primers (sll0832-3 '; SEQ ID NO: 4) amplified.

Primer sll0832-5 '

5'-GGATCCATGGGACGTTGGCCCACTGG-3 '(SEQ ID NO: 3)

Primer sll0832-3 '

5'-GTCGACCTAGCAACGGCCGCTATCC-3 '(SEQ ID NO: 4)

The PCR was carried out in a 50 µl reaction mixture which contained:
5 µl of a Synechocystis spec. PCC 6803 cell suspension
0.2 mM dATP, dTTP, dGTP, dCTP
1.5 mM Mg (OAc) ₂
5 µg bovine serum albumin
40 µmol oligonucleotide primer sll0832-5 '
40 µmol oligonucleotide primer sll0832-3 '
15 µl 3.3X rTth DNA Polymerase XL buffer (PE Applied Biosystems)
5U rTth DNA Polymerase XL (PE Applied Biosystems)

The PCR was carried out under the following cycle conditions:
Step 1: 5 minutes 94 ° C (denaturation)
Step 2: 3 seconds at 94 ° C
Step 3: 1 minute 52 ° C (annealing)
Step 4: 1 minute 72 ° C (elongation)
35 repetitions of steps 2 to 4
Step 5: 10 minutes 72 ° C (post-elongation)
Step 6: 4 ° C (holding pattern)

The amplificate (468 base pairs) was cloned into the PCR cloning vector pCR-Script (Stratagene) using standard methods. The construct is called pCR-Script / sll0832. The identity of the amplicon generated was confirmed by sequencing using the T3 and T7 primers (cf. SEQ ID NO: 1). Fig. 1 illustrates pCR-Script / sll0832 is, where "A" coding for Tocen-1 DNA fragment (456 base pairs) representiert.

Example 2 Generation of a Tocen-1 knock out mutant

A DNA construct for generating a deletion mutant of Tocen-1 in Synechocystis spec. PCC 6803 was created using standard cloning techniques. The vector pCR-Script / sll0832 was partially digested using the restriction enzyme NcoI and the protruding ends of the restriction digest were converted into blunt ends using standard methods. The aminoglycoside-3'phosphotransferase of the transposon Tn903 was cloned into the blunt-ended NcoI site of the open reading frame of Tocen-1. For this purpose, the Tn903 was isolated as an EcoRI fragment from the vector pUC4K (Vieira J and Messing J (1982) Gene 19: 259-268), the protruding ends of the restriction digest were converted into blunt ends according to standard methods and cut into the NcoI vector pCR-Script / sll0832 ligated (Ncol with smooth ends). The ligation approach was used to transform E.coli X11 blue cells. Transformants were selected using kanamycin and ampicillin. A recombinant plasmid (pCR-Script / sll0832 :: tn903; see FIG. 2) was isolated and used to transform Synechocystis spec. PCC 6803 used according to the Williams method (Williams (1987) Methods Enzymol 167: 776-778).

The Kanamycin cassette of the tn903 inserted in pCR-Script / sll0832 such that the transcription of the kanamycin resistance gene of the Tn903 in the opposite direction to the transcription of the open Tocen-1 reading frame runs.

In Figure 2, fragment A '(268 base pairs) contains the 5' region of Tocen-1, fragment B the transposon 903 (1286 base pairs) and fragment 'A (194 base pairs) the 3' region of Tocen-1.

Synechocystis spec. PCC 6803 transformants were selected on Kanamycin-containing (km) BG-11 solid medium (Castenholz (1988) Methods in Enzymology page 68-93) at 28 ° C. and 30 μmol photons * (m ² .s) ^-1 . Eight independent knockout mutants were generated after five rounds of selection (passages from individual colonies to fresh BG-11 km medium). The complete loss of the Tocen-1 endogen or the exchange for the recombinant Tocen-1 :: tn903 DNA was confirmed by PCR analysis.

Example 3 Tocopherol production in Synechocystis spec. PCC 6803 Wild-type cells and the Tocen-1 knockout mutants

The cells, each cultured on the BG-11 km agar medium, from two independent Synechocystis spec, PCC 6803 Tocen-1 knockout mutants and untransformed wild-type cells (on BG11 agar medium without kanamycin) were used to inoculate liquid cultures. For this purpose, cells of the mutant or the wild type Synechocystis spec. Transfer PCC 6803 from plate to 10 ml liquid culture. These cultures were cultivated at 28 ° C. and 30 μmol photons. (M ² .s) ^-1 (30 μE) for about 3 days. After determining the OD _{730 of} the individual cultures, the OD _{730 of} all cultures was synchronized by appropriate dilutions with BG-11 (wild types) or BG-11 km (mutants). These cultures, synchronized to cell density, were used to inoculate three cultures of the mutant or the wild-type control. The biochemical analyzes could thus be carried out using three independently grown cultures of a mutant and the corresponding wild types. The cultures were grown to an optical density of OD ₇₃₀ = 0.3.

The medium of the cell culture was obtained by centrifugation twice at 14000 rpm in an Eppendorf table centrifuge. The then disrupting the cells and extracting the Tocopherols and tocotrienols were incubated twice in an Eppendorf shaker at 30 ° C, 1000 rpm in 100% methanol for Minutes, combining the supernatants obtained in each case were. No further incubation steps resulted Release of tocopherols or tocotrienols.

In order to avoid oxidation, the extracts obtained were direct after extraction using a Waters Allience 2690 HPLC Plant analyzed. Tocopherols and tocotrienols were added via a Reverse phase column (ProntoSil 200-3-C30, Bischoff) with one mobile phase separated from 100% methanol and based on Standards (Merck) identified. Served as a detection system the fluorescence of the substances (excitation 295 nm, emission 320 nm) using a Jasco FP 920 fluorescence detector was proven.

The Tocen-1 knockout mutants surprisingly showed in comparison to the Synechocystis spec. PCC 6803 wild-type cells lost tocopherol and tocotrienol production ( Fig. 3).

Example 4 Manipulation of tocopherol biosynthesis in Nicotiana tabacum Samsun NN, Arabidopsis thaliana and Brassica napus by transgenic expression of Tocen-1

Transgenic plants are produced, the Tocen-1 on the one hand Control of the constitutive promoter of nitrilase-1 (nit1) A. thaliana gene (GenBank Acc.-No .: Y07648.2, nucleotides 2456-4340, Hillebrand et al. (1996) Gene 170: 197-200) and at others under the control of the USP's seed-specific promoter (unknown seed protein) gene from Vicia faba (Bäumlein et. al. (1991) Mol Gen Genet 225: 459-467). Furthermore Tocen-1 is used as a fusion protein with the transit peptide of rbcS- Proteins (Guerineau et al. (1988) Nucl Acid Res 16: 11380) both expressed both constitutively and seed-specifically. As Expression vectors serve derivatives of the pSUN vector (WO 02/00900).

The vector pSUN2 was used for the constitutive expression Use of standard methods changed so that the ocs- Terminator (Gielen et al. (1984) EMBO J 3: 835-846) as SalI- PacI-ocs-HindIII fragment (Sequence ID No: 5) is introduced. By introducing a linker, an additional SwaI Interface introduced in the polylinker (SEQ ID No: 6 and SEQ ID No: 7). The resulting plasmid is made with pSUN2-1 designated (Sequence ID NO: 8).

For expression as a fusion protein with the rbcS transit peptide the DNA fragment is coding for the rbcS transit peptide as SacI-SwaI fragment amplified (Sequence ID No: 9) and in the pSUN2-1 introduced. The vector is called pSUN2-2 (Sequence ID NO: 10).

For the expression of Tocen-1 under constitutive control the ORF as a SwaI-PacI fragment using standard methods using the primers 0832ex1-5 'and 0832ex1-3' (SEQ ID No. 11 and SEQ ID No. 12) amplified. The resulting 462 bp fragment becomes in the pCR4Blunt-TOPO (Invitrogen) according to the manufacturer cloned and sequenced using the T3 and T7 primers.

A correct clone is cut with SwaI-PacI and ligated into a SwaI-PacI cut pSUN2-1 using standard methods. In the resulting construct, which is cut with Ecl136II, the nitrilase promoter is cloned as an XhoI fragment (SEQ ID No: 13), the overhanging ends of which are filled in with Klenow polymerase according to standard methods. This plasmid is designated pSUN2 / NitP / sll0832 / ocs (SEQ ID No: 14) and used to generate transgenic plants (cf. FIG. 4).

To construct a plasmid that expresses Tocen-1 as a fusion protein with the transit peptide of the rbcS protein under the control of the constitutive promoter NitP, pSUN2-2 is cut with SwaI and PacI. The vector cut in this way is ligated with the Tocen-1 fragment, which is isolated from pCR-Script / sll0832 after digestion with SwaI and PacI. The nitrilase promoter (SEQ ID NO: 13) as the XhoI fragment, the overhanging ends of which are filled in with Klenow polymerase according to standard methods, is ligated into the resulting plasmid, which is cut with Ecl136II. This cloning allows the expression of a fusion protein which contains the transit peptide of the rbcs protein and Tocen-1 (see FIG. 5, SEQ ID No. 15).

To generate a plasmid which is the seed-specific Expression of Tocen-1 in plants is made possible seed-specific promoter of the USP gene from Vici faba used, which is present as an EcoRI-NcoI fragment in a pUC derivative (SEQ ID NO: 16).

For the cloning under the control of the USP promoter, the open reading frame of Tocen-1 is amplified using standard PCR methods in such a way that a BbsI interface is generated at the 5 'end and an EcoRV interface at the 3' end , The following oligonucleotide primers are used for this:
sll0832ex2-5 ': (SEQ ID NO: 17) and
sll0832ex2-3 ': (SEQ ID No: 18)

The amplificate is cloned in pCR4Blunt-TOPO and sequenced. The PCR product is ligated as a BbsI-EcoRV fragment in the vector cut with NcoI (overhanging ends compatible with 5 'overhang of the PCR product) and EcoRV. The expression construct is ligated as a PmeI and AfeI fragment in the pSUN2 cut with EcoRV. The resulting plasmid is called pSun2-USP-sll0832-3'cat (see FIG. 6, SEQ ID NO: 19).

For the construction of a plasmid that allows seed-specific expression as a fusion protein with the rbcS transit peptide, the rbcS fragment is amplified together with the ocs terminator as an NcoI-HindIII fragment (SEQ ID No: 20) from pSUN2-2 and into the Cloned pUC derivative containing the USP promoter. Promoters, rbcS and ocs are cloned as PmeI-AfeI fragments in pSUN2 cut with EcoRV. Tocen-1 is cloned into this expression vector as a SwaI-PacI fragment. The resulting plasmid is called pSUN2 / USP / rbcS / sll0832 / ocs ( Fig. 7, SEQ ID No: 21).

Example 5 Production of transgenic A.thaliana plants

Wild type A.thaliana plants (Columbia) are grown with the Agrobacterium tumefaciens strain (EHA105) based on a modified method (Steve Clough and Andrew Bent (1998) Plans J 16 (6): 735-743) using the vacuum infiltration method Bechtold et al. (Bechtold N et al. (1993) CRAcad Sci Paris 1144 (2): 204-212).

The A.tumefaciens cells are used in advance with the Plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSUN2 / USP / sll0832 / catT (SEQ ID NO: 19) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21) transformed.

Seeds of the Agrobacterium-transformed primary transformants are selected based on antibiotic resistance. Antibiotic resistant seedlings are planted in soil and used as fully developed plants for biochemical analysis.

Example 6 Production of transgenic Brassica napus plants

The production of transgenic oilseed rape plants is based on one Protocol from Bade JB and Damm B (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38), in which also the Composition of the media and buffers used is specified.

The transformations are carried out with the Agrobacterium tumefaciens Strains EHA105 or GV3101. For transformation, the Plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSUN2 / USP / sll0832 / catT (SEQ ID NO: 19) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21) used.

Seeds of Brassica napus var. Westar are made with 70% ethanol (v / v) Surface sterilized, 10 minutes at 55 ° C in water washed, in 1% hypochlorite solution (25% v / v Teepol, 0.1% v / v Tween 20) incubated for 20 minutes and six times with sterile Washed water for 20 minutes each. The seeds are three Days dried on filter paper and 10 to 15 seeds in one Glass flask germinated with 15 ml germination medium. Of the roots and several seedlings (approx. 10 cm tall) Apices removed and the remaining hypocotyls in about 6 mm cut long pieces. The approximately 600 explants obtained in this way are washed for 30 minutes with 50 ml of basal medium and in a 300 ml flask transferred. After adding 100 ml The callus induction medium was grown at 100 rpm for 24 hours incubated.

From the individual with the plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pStJN2 / USP / sll0832 / catT (SEQ ID NO: 19) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21) transformed Agrobacteria strains, an overnight culture at 29 ° C. is prepared in Luria Broth medium with kanamycin (20 mg / l), of which 2 ml in Incubate 50 ml Luria Broth medium without kanamycin for 4 hours at 29 ° C up to an OD ₆₀₀ of 0.4 to 0.5. After pelleting the culture at 2000 rpm for 25 min, the cell pellet is resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution is adjusted to an OD ₆₀₀ of 0.3 by adding further basal medium.

The rapeseed explants become the callus induction medium sterile pipettes removed, 50 ml Agrobacterium solution added, mixed gently and incubated for 20 min. The Agrobacteria suspension is removed, the rape explants for 1 min washed with 50 ml callus induction medium and then 100 ml callus induction medium added. The co-cultivation is on a rotary shaker at 100 rpm for 24 h carried out. The co-cultivation is done by removing the callus Induction medium stopped and the explant twice for 1 20 min with 25 ml and twice for 60 min with 100 ml each Wash medium washed at 100 rpm. The washing medium with the explants is transferred to 15 cm petri dishes and the medium with sterile Pipettes removed.

20 to 30 explants in 90 mm are used for regeneration Petri dishes transferred containing 25 ml sprout induction medium Kanamycin included. The Petri dishes are made with 2 layers of leucopor closed and at 25 ° C and 2000 lux with photoperiods of Incubated 16 hours light / 8 hours dark. Every 12 days the developing calli on fresh petri dishes Sprout induction medium transferred. All further steps to Regeneration of entire plants is carried out as by Bade JB and Damm B (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995, 30-38) described.

Example 7 Production of transgenic Nicotiana tabacum plants

10 ml of YEB medium with antibiotic (5 g / l beef extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose and 2 mM MgSO _4. ) With a colony of the individual with the Plasmids pSUN2 / NitP / sll0832 / ocs (SEQ ID NO: 14), pSUN2 / NitP / rbcS / sll0832 / ocs (SEQ ID NO: 15), pSUN2 / USP / sll0832 / catT (SEQ ID NO: 19) or pSUN2 / USP / rbcS / sll0832 / ocs (SEQ ID NO: 21) inoculated transformed Agrobacterium tumefaciens strains and cultured overnight at 28 ° C. The cells are pelleted in a table centrifuge for 20 min at 4 ° C., 3500 rpm and then resuspended in fresh YEB medium without antibiotics under sterile conditions. The cell suspension is used for the transformation.

The wild-type plants from sterile culture are obtained by vegetative replication. For this purpose, only the tip of the plant is cut off and transferred to fresh 2MS medium in a sterile mason jar. The hair on the top of the leaf and the central ribs of the leaves are removed from the rest of the plant. The leaves are cut into approximately 1 cm ² large pieces with a razor blade. The agrobacterial culture is transferred to a small petri dish (diameter 2 cm). The leaf pieces are briefly drawn through this solution and placed with the underside of the leaf on 2MS medium in Petri dishes (diameter 9 cm), so that they touched the medium. After two days in the dark at 25 ° C, the explants are transferred to plates with callus induction medium and heated to 28 ° C in the climatic chamber. The medium must be changed every 7 to days. As soon as calli were formed, the explants were placed in sterile mason jars on shoot induction medium with Claforan (0.6% BiTec agar (w / v), 2.0 mg / l zeatin ribose, 0.02 mg / l naphthylacetic acid, 0.02 mg / l Gibberelic acid, 0.25 g / ml claforan, 1.6% glucose (g / v) and 50 mg / l kanamycin). After about a month, organogenesis occurs and the shoots formed can be cut off. The shoots are cultivated on 2MS medium with Claforan and a selection marker. As soon as a strong root ball has formed, the plants can be potted in prickly potting soil.

Example 8 Characterization of the transgenic plants

In order to confirm that the expression of Tocen-1 affects the vitamin E biosynthesis in the transgenic plants, the tocopherol and tocotrienol contents in leaves and seeds of the plants transformed with the described constructs (Arabidopsis thaliana, Brassica napus and Nicotiana tabacum) analyzed. For this purpose, the transgenic plants are cultivated in the greenhouse and plants which express the gene coding for Tocen-1 are identified at the Northern level. The tocopherol content and the tocotrienol content are determined in the leaves and seeds of these plants. In all cases, the tocopherol or tocotrienol concentration is increased compared to non-transformed plants. SEQUENCE LISTING

Claims (9)

1. A method for increasing the vitamin E content in a plant organism or a tissue, organ, part, cell or propagation material thereof, comprising a) transgenic expression of a protein with the SEQ ID NO: 2 or a functional equivalent thereof with an identity of at least 60% to the sequence with the SEQ ID NO: 2, and b) Selection of plant organisms in which - in contrast to or compared to the parent organism - the vitamin E content in said plant organism or in a tissue, organ, part, cell or propagation material of the same is increased. 2. The method of claim 1, wherein the plant is an oil plant is. 3. Transgenic expression construct fully under control one in a plant organism or tissue, Organ, part or cell of the same functional promoter a nucleic acid sequence coding for a protein with the SEQ ID NO: 2 or a functional equivalent thereof with at least 60% identity to the sequence the SEQ ID NO: 2. 4. Transgenic expression construct according to claim 3, wherein the nucleic acid sequence is described by a) a sequence with SEQ ID NO: 1 or b) a sequence which is derived from a sequence with SEQ ID NO: 1 in accordance with the degenerate genetic code, or c) a sequence which is at least 60% identical to the sequence with SEQ ID NO: 1. 5. Transgenic expression construct according to one of claims 3 or 4, the promoter being a constitutive or a seed-specific promoter. 6. Transgenic expression vector containing a transgenic Expression construct according to one of claims 3 to 5. 7. Transgenic plant organism or tissue, organ, part, Cell or propagation material of the same, containing one transgenic expression construct according to any one of claims 3 to 5 or a transgenic expression vector according to claim 6. 8. Transgenic plant organism according to claim 7, wherein the plant organism is selected from the group of oil plants consisting of Borago officinalis, Brassica campestris, brassica napus, brassica rapa, cannabis sativa, Curthamus tinctorius, Cocos nucifera, Crambe abyssinica, Cuphea species, Elaeis guinensis, Ekeis oleiferu, Glycine max, Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum, Helianthus annus, Linum usitatissimum, Oenethern biennis, Ozea europea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticum species, Zea maize, and walnut Almond. 9. Use of a transgenic plant organism or Tissue, organ, part, cell or propagation material of the same one of claims 8 or 9 for the production of vitamin E, Oils, fats, free fatty acids or derivatives of foregoing.